Abstract

Parkinson’s disease (PD) is the commonest neurodegenerative cause of parkinsonism and, in terms of prevalence, is the fastest growing neurological disorder in the world. In the clinical setting, there are challenges in achieving an early and accurate diagnosis of PD due to clinical overlap with less common parkinsonian disorders, such as progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA).

Furthermore, there is variability in disease progression both between and within PD, PSP, CBS, DLB and MSA such that it can be challenging to give patients accurate prognostic information. The combination of diagnostic uncertainty and varying rates of progression also impact on clinical trials which rely on studying homogenous disease groups.

In this article we will review the current state of biofluid biomarker development and its potential applications to Parkinsonian disorders, the challenges and pitfalls of these techniques, and future directions.

Introduction

In the UK, approximately 150,000 people are living with Parkinson’s disease (PD) and this is estimated to rise to around 172,000 people by 2030 [1]. Parkinsonian disorders include PD and the Parkinson-plus syndromes, progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Each of these disorders are associated with the presence of distinct structures of pathologically misfolded protein at post-mortem. The self-templating properties of these protein pathologies mean that they can induce protein misfolding in neighbouring cells. This then leads to cell-to-cell spread of the pathology (referred to as prion-like seeding) through distinct regions of the brain leading to associated neurodegeneration and progressive motor and cognitive impairments [2], and there are no effective disease-modifying therapies for these disorders currently.

The primary neuropathology of PD, DLB and MSA is characterised by a build-up of pathologically misfolded forms of alpha-synuclein (a-syn) as neuronal Lewy bodies (in PD and DLB) or glial cytoplasmic inclusions (in MSA) [3]. Of note, Lewy bodies can also be found as a secondary co-pathology in other neurodegenerative disorders. In contrast, PSP and corticobasal degeneration (CBD) are associated with a build-up of pathologically misfolded 4-repeat tau (4RT) in neurons and astrocytes, and this hardly ever occurs as a co-pathology in other disorders [3].

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Achieving an early and accurate clinical diagnosis in patients presenting with symptoms suggestive of an underlying Parkinsonian disorder is challenging. This is especially true in the early stage of symptomatic disease when there is significant clinical overlap between PD and the Parkinson-plus disorders. This diagnostic challenge has been highlighted in previous longitudinal cohort studies of patients who fulfil PD clinical diagnostic criteria where up to 10% will go on to have a revised diagnosis [4]. In the same vein, we have shown that delays in diagnosis are significant in PSP, with the diagnostic latency being up to 4 years [5]. Furthermore, clinical features are limited in their ability to predict the rate of disease progression which is variable both between and within the Parkinsonian disorders [5].

Therefore, in the absence of objective diagnostic and prognostic biomarkers, relying on a clinical assessment alone frequently leads to misdiagnosis, delayed diagnosis, and unpredictable prognosis in the parkinsonian disorders. This in turn leads to diagnostic uncertainty for patients and a lack of homogenous cohorts for disease-modifying clinical trials which may, in part, explain why a-syn and tau-targeting immunotherapies have so far failed to show disease modification in PD and PSP clinical trials [6,7]. For these reasons, there is an increasing focus on the development of diagnostic and prognostic biofluid biomarkers.

Measurements of alpha-synuclein and tau

Many studies have explored whether there are alterations in blood and CSF levels of total and/or phosphorylated a-syn in PD and MSA and tau in PSP relative to age-matched controls. Initial reports of lower CSF total a-syn in PD and MSA vs. PSP and controls [8] were hypothesised to reflect ‘pathological protein trapping’ within aggregates, much like in Alzheimer’s disease (AD) where reduced levels of Aβ42 are a marker of amyloid plaque pathology. However, this finding was not consistently reproducible in subsequent studies and so its diagnostic value remains inconclusive [9]. A more promising approach may lie in the measurement of oligomers as the early toxic a-syn species in the synucleinopathy disorders. Initial studies demonstrated significantly elevated CSF levels of oligomeric a-syn and the oligomer/total a-syn ratio in PD patients vs. controls and PSP patients [10]. Furthermore, longitudinal measures of CSF oligomeric a-syn have been shown to track clinical disease progression suggesting prognostic value [11].

In contrast to the robust finding of elevated total and phosphorylated species of tau in blood and CSF of AD patients, this has not been demonstrated in PSP or CBD [8]. As such, there is ongoing work to identify measurable PSP and CBD-specific tau epitopes that may potentially represent disease-specific biomarkers for the 4-repeat tauopathies.

Biomarkers of neuroinflammation

Alongside the primary pathologies of misfolded alpha-synuclein and tau, there has been increasing interest in the role of concomitant neuroinflammation as a pathological process across the neurodegenerative Parkinsonian disorders, mediated by microglia and astrocytes. Furthermore, neurotoxicity and subsequent disease progression may be determined by the balance between proinflammatory and neuroprotective states of these glial cells, driven by the release of certain inflammatory cytokines such as interleukins and tumour necrosis factor, suggesting that neuroinflammation may be a disease-modifying target [12]. Recently, isolated studies using novel multiplex blood-based proteomics platforms such as Olink and NULISA have highlighted distinct neuroinflammatory biomarker signatures in PD and PSP from both a diagnostic and prognostic perspective, although independent replication is currently lacking [13,14].

Neurofilament light chain

Neurofilament light chain (NFL) is a marker of axonal degeneration. Unsurprisingly, it is elevated at varying levels in both blood and CSF across many neurodegenerative diseases including the Parkinsonian disorders [15]. Therefore, NFL is not specific to an underlying neurodegenerative pathology and has limited diagnostic value. However, the prognostic value of NFL has been highlighted in multiple studies across PD, PSP and MSA where baseline levels have been shown to predict the rate of clinical decline [16-18].

Seed amplification assays

There has been recent interest in the development of seed amplification assays (SAA) which detect seeding-competent aggregates, via the measurement of Thioflavin T fluorescence, as a marker of misfolded protein pathology (Figure 1). Assay development has mainly focused on a-syn SAA which has been shown to reliably differentiate PD from healthy controls with high sensitivity and specificity. A-syn SAA may also be reliable in detecting pathology in the prodromal phase of PD and in differentiating PD and DLB from MSA [19].

Figure 1: Seed amplification assay aggregation curve. Dashed horizontal line indicates threshold for determining assay positivity. Associated quantitative seeding kinetic measures highlighted: lag phase or time to threshold; maximum ThT fluorescence signal (ThT max); area under the curve (AUC).  

As such, there are growing calls to formalise a biological definition of neuronal a-syn disease based on a-syn SAA positivity for future research studies [20,21]. This is similar to the AD International Working Group who define AD as a clinical-biological entity where diagnosis is made in consideration of both a clinical disorder and the support of positive amyloid and tau biomarkers [22]. It is also noteworthy that a-syn SAA is positive in approximately 5-15% of age-matched controls and so, in line with a clinical-biological definition, these individuals should be considered “at risk” of developing PD, DLB or MSA.

Additionally, our own recent work has shown that the a-syn SAA is also positive in a subset of clinically diagnosed PSP CSF samples, likely due to the presence of Lewy body co-pathology, adding further complexity to the interpretation of SAA results [23]. Therefore, using a-syn SAA in isolation cannot reliably differentiate between PD and PSP. As such, we have been developing a 4RT SAA which we propose to use in combination with a-syn SAA for more accurate diagnostic differentiation between PD, DLB, MSA and PSP.

Furthermore, in our study of over 800 PD participants in the Parkinson’s Progression Markers Initiative, we have highlighted the potential prognostic value of a-syn SAA by showing that the quantitative seeding kinetic measure, time to threshold, at baseline predicts subsequent cognitive decline, potentially due to faster seeding kinetics increasing the rate of cortical Lewy body spread [24] (Figure 2).

Figure 2: Time-to-event (cognitive decline) analysis stratified by baseline CSF a-syn SAA time to threshold (TTT). Cognitive decline defined as reaching MoCA ≤21 during the study period. Fast TTT = samples that were within the bottom quartile of TTT values; Slow TTT = samples that were within the top three quartiles of TTT values. Cox-proportional hazards model, adjusting for sex, age and disease duration at baseline produced a p-value of 0.001.

DOPA decarboxylase

DOPA decarboxylase (DDC) is an emerging biomarker of interest. In the physiological state, DDC has an important role in the production of neurotransmitters in the brain, and it is also essential to the formation of dopamine from exogenous levodopa treatment. Recent studies have shown raised plasma and CSF DDC levels in PD, PSP, CBS and MSA relative to controls, as well as in drug-naive PD patients. Of note, compared to patients with de novo PD, the medicated PD group showed higher DDC levels. Interestingly, DDC levels were also raised in at risk preclinical patients, i.e. neurologically normal controls who are a-syn SAA positive [25]. It is hypothesised that raised DDC levels are a compensatory mechanism in the hypodopaminergic state seen across the Parkinsonian disorders, although further studies are needed to explore this as well as the potential of DDC as an early diagnostic biomarker.

Conclusions and future directions

There is a need for objective biomarkers for the Parkinsonian disorders to enable early and accurate diagnosis and prognosis in clinical practice. The emergence of a-syn SAA has been a significant development in this field and has shown diagnostic and prognostic potential in PD. However, co-pathologies and incidental pathologies are potential pitfalls in the interpretation of SAA results and highlight the fact that there is no such thing as a perfect biomarker. Instead, the focus should be on achieving a biomarker-supported diagnosis that does not devalue the contribution of clinical features, which has been one of the concerns of those opposing the proposed biological definitions of PD.

There is great potential in the combination of a-syn and 4RT SAA testing to enhance biomarker-supported differentiation between the Parkinsonian disorders. Furthermore, future multi-modal approaches of combining clinical features with biofluid, imaging and digital biomarkers may provide even greater diagnostic and prognostic power. Finally, it is likely that SAA and its associated kinetic measures will refine the design of future clinical trials as a patient inclusion and stratification tool, and potentially as a surrogate marker of treatment response.

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